Hyaluronan (HA), a secreted polymeric glycosaminoglycan component of extra cellular matrices, is essential for cell growth and migration during normal development. Elevated production of HA is associated with several pathological states, including cancer. Studies of human prostate tumor sections have demonstrated that HA, not present in normal adult prostate, is dramatically overproduced as a function of disease severity in prostate cancer patients. Tumor cell associated HA is observed in the most invasive cancers and correlates to a worse prognosis for the patient. Overproduction and manipulation of cell surface HA is regulated by the action of hyaluronan synthase enzymes (HAS) and hyaluronidases. Degradation of HA by hyaluronidases produces small oligosaccharide fragments, which are active angiogenic stimuli. The long-term goals of the laboratory are to understand how HA synthases and hyaluronidases function in concert to facilitate tumor progression in a physiologically relevant model system. The following specific aims are proposed. Aim 1: Investigate how the balance between HA synthase and hyaluronidase activities regulates the production of biologically active HA. These enzymes will be expressed in prostate tumor cell lines, the resulting quantity and size of HA produced by the cells will be quantified, and the tumorigenic potential of the cells determined by orthotopic injection in mice. Aim 2: Use an inducible system to evaluate the role of HA in tumor growth, regression, and apoptosis. Tetracycline inducible constructs will be used to up regulate HA synthesis in non-tumorigenic prostate tumor cells and to inhibit HA synthesis in highly aggressive prostate tumor cells in an experimentally controlled fashion. In addition, the chronological importance of HA in tumorigenesis and its mode of action will be evaluated by eliminating HA production in an established orthotopic tumor and determining vascular density and relative percentages of proliferating and apoptotic cells in tumor sections. Aim 3: Assess the contribution of selected HA binding proteins to HA biological activity. Since HA is a secreted product, its maintenance in stromal and pericellular matrices is dependent upon auxiliary binding proteins. This aim will investigate the role of specific HA binding proteins in production and retention of HA at the cell surface, determine whether these HA binding proteins contribute to prostate tumorigenesis, and evaluate direct interaction with HA metabolic enzymes.